Int J Clin Exp Pathol 2015;8(9):11766-11771 www.ijcep.com /ISSN:1936-2625/IJCEP0012638
Case Report A case of gastric cancer with heterogeneous components of EB virus (+)/TP53 (+) and EB virus (-)/TP53 (-) Ikuo Matsuda1, Kazuomi Kan2, Sadayuki Doi2, Yoshiyuki Motoki2, Masayuki Onodera3, Seiichi Hirota1 1 3
Department of Surgical Pathology, Hyogo, Japan; 2Department of Surgery, Kawanishi City Hospital, Hyogo, Japan; Department of Pathology, Kawanishi City Hospital, Hyogo, Japan
Received July 9, 2015; Accepted August 21, 2015; Epub September 1, 2015; Published September 15, 2015 Abstract: Epstein-Barr virus (EBV)-associated gastric adenocarcinoma is a histological subtype of gastric adenocarcinoma, in which all of the carcinoma cells are basically positive for EBV-encoded small RNA (EBER) by in situ hybridization. Although its typical histology has some overlap with gastric carcinoma with lymphoid stroma, absence of massive lymphoid infiltrate is sometimes observed either in whole or in part. EBV-associated adenocarcinoma is one of the four representative molecular pathological subtypes recently identified by comprehensive genomic analysis of gastric adenocarcinomas. According to the analysis, typical EBV-associated gastric adenocarcinoma constitutes an independent molecular pathological subgroup, which is mutually exclusive to TP53-mutated adenocarcinoma with chromosomal instability, another molecular pathological subtype in gastric adenocarcinomas. Here, we report a rare case of gastric cancer heterogeneously composed of EBER (+)/TP53 (+) and EBER (-)/TP53 (-) portions. The EBER (+)/TP53 (+) component with massive lymphoid infiltrate surrounded the EBER (-)/TP53 (-) component showing well to moderately differentiated tubular adenocarcinoma. Although collision of two independent gastric cancers could be the simplest and most possible explanation for this situation, we discussed another possibility. In the case of gastric collision tumors, concurrent development of EBER (+) gastric adenocarcinomas and EBER (-) gastric adenocarcinomas in a single stomach is a rare incident. Since presence of the EBER (+)/TP53 (+) tumor component is atypical in itself, we also discussed the mechanism of development of the clone. Keywords: Gastric adenocarcinoma, Epstein-Barr virus, adenocarcinoma with lymphoid stroma, TP53, collision tumor
Epstein-Barr virus (EBV)-associated gastric adenocarcinoma is a histological subtype of gastric adenocarcinoma, in which all of the carcinoma cells are basically positive for EBVencoded small RNA (EBER) by in situ hybridization [1]. Therefore, it is reasonable to assume that the EBV infection occurs at the early stage of the tumorigenesis and may be related to its initiation. Although its typical histology has some overlap with gastric carcinoma with lymphoid stroma, absence of massive lymphoid infiltrate is sometimes observed either in whole or in part. More than 80% of cases of gastric carcinoma with lymphoid stroma are associated with Epstein-Barr virus (EBV) infection [2].
molecular pathological subtypes [3]. The first subtype is EBV-associated adenocarcinoma, the second one is associated with microsatellite instability characterized by loss of expression of MLH1 protein, the third one is diffuseproliferating and poorly-differentiated histological subtype characterized by mutations in RhoA signaling pathway, and the last one is intestinal subtype often associated with chromosomal instability and TP53 mutation [3]. The subtype with MLH1 loss is often associated with CpG island methylator phenotype [3]. It was reported that these four subtypes are mutually exclusive in general [3]. Therefore, EBER mRNA positivity concurrent with diffuse TP53 protein positivity within a tumor is considered to be an exception rather than a rule.
A recent comprehensive genomic analysis of gastric adenocarcinoma identified at least four
Histologically, it is common that mixture of different patterns composes a tumor. At cellular
Introduction
Gastric cancer with EBV (+)/TP53 (+) and EBV (-)/TP53 (-) components TP53 (+) component in the peripheral zone surrounded EBER (-)/TP53 (-) component in the central zone. Although we considered that collision of two independent gastric cancers could be the most possible explanation for this situation, we discussed another possibility. Since EBER (+)/TP53 (+) tumor component in this tumor is atypical in itself, we discussed mechanism for this phenomenon. Case report
Figure 1. Loupe view of the resected tumor reconstructed by two virtual slides. (A) Hematoxylin-eosin (HE) stain. (B) In situ hybridization of Epstein-Barr virus-encoded small RNAs (EBER). (C) TP53 immunohistochemistry. The area of the whole tumor is outlined by a dotted curve in (A). In (B) and (C), positive cells are stained brown. The intratumor heterogeneity was illustrated by in situ hybridization of EBER (B) and immunohistochemistry for TP53 (C). That is, the tumor cells in the peripheral zone, demarcated by a solid curve in each figure, were positive for EBER (B) as well as TP53 (C). In contrast, the tumor cells in the central zone, which is the residual portion of the tumor and is demarcated by dotted and solid lines, were basically negative for EBER (B) and TP53 (C). Both of the two zones were homogeneously positive for MLH1 immunohistochemistry (data not shown). Bar: 5 mm.
level, moreover, tumors are composed of assembly of multiple heterogeneous clones. The so-called intratumor heterogeneity has been recently refined as genomic heterogeneity among the tumor cells, revealed by analysis using next generation sequencing [4-6]. The case of gastric adenocarcinoma is no exception. In the routine diagnostic histopathology, the correlation between morphological heterogeneity and genomic one in the tumor is of clinical significance. Morphological heterogeneity of the tumor may sometimes provide an important clue to genomic heterogeneity of the tumor, which is often associated with responses to particular molecular-targeted therapy. A collision tumor, in which more than two independent tumors collide to constitute a tumor, might be one of the extreme presentations of intratumor heterogeneity. Here we report a rare case of gastric cancer heterogeneously composed of EBER (+)/TP53 (+) and EBER (-)/TP53 (-) portions. EBER (+)/
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A 70-year-old Japanese man was admitted to Kawanishi City Hospital for examination of progressive anemia. At the age of 58, he had suffered from acute myocardial infarction, for which coronary stenting was performed, and intrahepatic cholangiocellular carcinoma, which was completely resected by partial hepatectomy. He had also been followed for type 2 diabetes mellitus and chronic hepatitis C. Blood test showed that his hemoglobin had decreased from 13.1 to 11.1 g/dL (normal range: 13.217.2) during 4 months. Gastrointestinal bleeding was suspected. Endoscopic examination of the gastrointestinal tract revealed Borrmann type 2 tumor of around 2.5 cm in diameter in the posterior wall of the gastric corpus (data not shown). The biopsy of the tumor showed the proliferation of tubular adenocarcinoma (data not shown). Pylorus-preserving partial gastrectomy was performed. The HE image of the resected specimen showed that the tumor was saucer-shaped at low power view (Figure 1A), which was composed of the central portion and the peripheral one. The deepest portion of the invasion reached to the superficial layer of the muscularis propria (Figure 1A). The central portion of the tumor mainly consisted of tubular proliferation of atypical epithelial cells, which was diagnosed as tubular adenocarcinoma of well to moderately differentiated type (Figure 2A). In contrast, peripheral portion consisted of tumor cells proliferating diffusely and sparsely, forming small aggregates or nests, which was diagnosed as poorly differentiated adenocarcinoma (Figure 2C). The morphological distinction between the central and peripheral components of the tumor was clearly highlighted with immunohistochemistry of pankeratin AE1/AE3 (Figures 2B, 2D, 3B). In addition to the morphological differences, the peripheral portion of
Int J Clin Exp Pathol 2015;8(9):11766-11771
Gastric cancer with EBV (+)/TP53 (+) and EBV (-)/TP53 (-) components
Figure 2. Representative histology of the resected tumor in the central and peripheral zones. (A, B) Central zone. (C, D) Peripheral zone. (A, C) HE stain. (B, D) Immunohistochemistry for pankeratin AE1/AE3. The tumor cells formed tubular structure in the central zone (A, B), while they proliferated diffusely in nested or isolated clusters intermingled with lymphoid infiltrate in the peripheral zone (C, D). Original magnification: × 200. Bar: 100 μm.
the tumor was intermingled with heavy lymphoid infiltrate, which obscured the distinction between the tumor foci and inflammatory cells (Figure 2C). This so-called lymphoepithelial lesion was reminiscent of EBV-associated carcinoma such as nasopharyngeal carcinoma. In order to examine a possibility of EBV-associated gastric adenocarcinoma, in situ hybridization of EBER was performed. Unexpectedly, the tumor cells were heterogeneously positive for EBER; that is, the central and tubular portion of the tumor was negative for EBER, while the peripheral and poorly differentiated portion of the tumor was positive (Figures 1B and 3C). Further immunohistochemical examination of the tumor showed that the central component of the tumor was almost negative for TP53 except for sparsely distributed positive cells in the mucosal surface, while the peripheral component was diffusely positive for TP53 (Figures 1C and 3D). Both the central and peripheral 11768
portions of the tumor were positively stained for MLH1 immunohistochemistry (Figure 3E). Thus, the central zone of the tumor was composed of EBER (-)/TP53 (-)/MLH1 (+) cells, while the peripheral zone of the tumor was composed of EBER (+)/TP53 (+)/MLH1 (+) cells (Figure 3C-E). Discussion In this paper, we report a case of gastric adenocarcinoma composed of two heterogeneous portions with clear borderline formation: the central core zone and the peripheral zone. The central zone was composed of EBER (-)/TP53 (-)/MLH1 (+) tumor cells. In contrast, the peripheral zone was composed of EBER (+)/TP53 (+)/ MLH1 (+) tumor cells, surrounding the central core zone. A recent comprehensive genomic analysis revealed that gastric adenocarcinomas are comInt J Clin Exp Pathol 2015;8(9):11766-11771
Gastric cancer with EBV (+)/TP53 (+) and EBV (-)/TP53 (-) components
Figure 3. High power view of the resected tumor at the borderline between the central and peripheral zones described in Figure 1. (A) HE stain. (B, D, E) Immunohistochemistry. (C) In situ hybridization. (B) Pankeratin AE1/AE3. (C) EBER. (D) TP53. (E) MLH1. In each figure, the central zone is in the left half side of the solid line, while the peripheral zone is in the right half side. In (B) to (E), positive cells are stained brown. All of the tumor cells were highlighted by AE1/AE3 immunohistochemistry (B). The central zone of the tumor in the left half of each figure was negative for EBER (C) and TP53 (D), and positive for MLH1 (E). On the other hand, the peripheral zone of the tumor in the right half of each figure was positive for EBER (C) and TP53 (D), as well as MLH1 (E). Original magnification: × 200. Bar: 100 μm.
posed of at least four molecular pathological subtypes, each characterized by association with either EBV, loss of MLH1 protein, TP53 mutation, or mutations in RhoA pathway [3]. It was reported that these four subtypes are mutually exclusive in general [3]. Consistent with this, there were several reports suggesting that EBV-associated gastric adenocarcinomas do not harbor TP53 mutations in most cases [7-11], although some discrepant results were also reported [12-14]. In this regard, the EBER (+)/TP53 (+) tumor component in the peripheral zone appears to be atypical for EBV-associated gastric adenocarcinoma. The most straightforward explanation for the present case might be a collision tumor composed of two independent gastric adenocarcinomas, one of which is EBER (+)/TP53 (+) while the other is EBER (-)/TP53 (-). EBV-associated gastric adenocarcinomas are reported to be associated with synchronous or metachronous independent adenocarcnomas in the stomach, and Matsunou et al. [15] reported that 22 out of 26 cases (86.4%) of gastric adenocarcinomas with lymphoid stroma which developed synchronously or metachronously in patients 11769
with gastric adenocarcinomas with lymphoid stroma were EBER-positive. They also described that all of 4 gastric adenocarcinomas without lymphoid stroma which developed synchronously or metachronously in patients with gastric adenocarcinomas with lymphoid stroma were EBER-positive as well [15]. Since these data indicate that concurrent development of EBER (-) gastric adenocarcinomas is a rare incident in the stomach with EBER (+) gastric adenocarcinomas, collision of EBER (+) adenocarcinoma with EBER (-) adenocarcinoma in our case might be an extraordinary event. As another possibility for the present case, one of the two zones may be a metastasis of other primary tumor to the original gastric cancer. Since the patient had undergone partial hepatectomy for cholangiocellular carcinoma 12 years before, the EBER (-)/TP53 (-)/MLH1 (+) zone could be the metastatic recurrence of the cholangiocellular carcinoma. Although the cholangiocellular carcinoma was also shown to be EBER (-)/TP53 (-)/MLH1 (+) (data not shown), CK7-positivity of the cholangiocellular carcinoma was not consistent with CK7-negativity of the present gastric cancer (data not shown). Int J Clin Exp Pathol 2015;8(9):11766-11771
Gastric cancer with EBV (+)/TP53 (+) and EBV (-)/TP53 (-) components How did TP53-immunopositivity evolved in EBER (+) tumor cells in the peripheral zone? A candidate cell-autonomous link, if any, between EBV infection and TP53 mutation may be the expression of apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) family proteins in the tumor cells [5, 16]. As a mechanism of host immunity to virus, APOBEC family restricts replication of viral genomes (for example, EBV genome) by enzymatically introducing cytidine to thymine conversion in the DNA [17]. This may not be achieved without the cost of potential introduction of mutations into the host genome, presumably including TP53 mutation. A recent study by next generation sequencing showed that intratumor heterogeneity is partly generated by an APOBEC-mediated mechanism [5]. Expression of activation-induced cytidine deaminase (AID), another APOBEC family, in gastric adenocarcinoma was also reported [18]. AID may be an APOBEC family involved in TP53 mutation in Helicobacter pylori-mediated tumorigenesis of the stomach [18]. The borderline formation between the two components in the present case can be easily explained by a cell competition mechanism at the junction of the two. A typical example of cell competition may be observed in senescenceassociated secretory phenotype (SASP) in aging, where senescent “loser” subclones cease to proliferate while the surrounding “winner” cells proliferate in response to cytokines secreted by the senescent “loser” subclones. It is tempting to speculate that cytokine secretion, presumably associated with inflammatory infiltration in EBV-positive cancer, may play a role in this borderline formation, just as in SASP. The importance of non-cell-autonomous driving of tumor growth and clonal heterogeneity has recently been experimentally substantiated [19]. In conclusion, we report a rare case of gastric cancer, in which EBER (-)/TP53 (-) component in the central zone and EBER (+)/TP53 (+) one in the peripheral zone are coexistent. A collision tumor of two independent gastric cancers could be the most possible explanation for this situation. Concurrent development of EBER (+) gastric adenocarcinoma and EBER (-) gastric adenocarcinoma in a single stomach is a rare incident. Moreover, EBER (+)/TP53 (+) tumor
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component in this tumor is considered to be atypical in itself for EBV-associated gastric adenocarcinoma. We speculate that the sharp borderline formation of two components in the present case may be promoted by cell-autonomous as well as non-cell-autonomous mechanisms, both of which may be related to EBV infection. Acknowledgements We thank all the colleagues in the Department of Surgical Pathology, Hyogo College of Medicine, and Department of Pathology, Kawanishi City Hospital, for preparation of pathological specimen. This work was partly supported by Grant-in-Aid for Researchers, Hyogo College of Medicine, 2014. Disclosure of conflict of interest None. Address correspondence to: Dr. Seiichi Hirota, Department of Surgical Pathology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 6638501, Japan. Tel: +81798456666; Fax: +81798456671; E-mail: [email protected]
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Shinozaki-Ushiku A, Kunita A, Fukayama M. Update on Epstein-Barr virus and gastric cancer (Review). Int J Oncol 2015; 46: 1421-34. Lauwers GY, Carneiro F, Graham DY, Curado MP, Franceschi S, Montgomery E, Tatematsu M, T Hattori T. Gastric carcinoma. In: Bosman FT, Carneiro F, Hruban RH, Theise ND, editors. World Health Organization Classification of Tumours of the Digestive System. 4th edition. Lyon: International Agency for Research on Cancer (IARC); 2010. pp. 48-58. Cancer Genome Atlas Research Network. Comprehensive molecular characterization of gastric adenocarcinoma. Nature 2014; 513: 2029. Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder D, Gronroos E, Martinez P, Matthews N, Stewart A, Tarpey P, Varela I, Phillimore B, Begum S, McDonald NQ, Butler A, Jones D, Raine K, Latimer C, Santos CR, Nohadani M, Eklund AC, Spencer-Dene B, Clark G, Pickering L, Stamp G, Gore M, Szallasi Z, Downward J, Futreal PA, Swanton C. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med 2012; 366: 883-892.
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de Bruin EC, McGranahan N, Mitter R, Salm M, Wedge DC, Yates L, Jamal-Hanjani M, Shafi S, Murugaesu N, Rowan AJ, Grönroos E, Muhammad MA, Horswell S, Gerlinger M, Varela I, Jones D, Marshall J, Voet T, Van Loo P, Rassl DM, Rintoul RC, Janes SM, Lee SM, Forster M, Ahmad T, Lawrence D, Falzon M, Capitanio A, Harkins TT, Lee CC, Tom W, Teefe E, Chen SC, Begum S, Rabinowitz A, Phillimore B, SpencerDene B, Stamp G, Szallasi Z, Matthews N, Stewart A, Campbell P, Swanton C. Spatial and temporal diversity in genomic instability processes defines lung cancer evolution. Science 2014; 346: 251-6. [6] Zhang J, Fujimoto J, Zhang J, Wedge DC, Song X, Zhang J, Seth S, Chow CW, Cao Y, Gumbs C, Gold KA, Kalhor N, Little L, Mahadeshwar H, Moran C, Protopopov A, Sun H, Tang J, Wu X, Ye Y, William WN, Lee JJ, Heymach JV, Hong WK, Swisher S, Wistuba II, Futreal PA. Intratumor heterogeneity in localized lung adenocarcinomas delineated by multiregion sequencing. Science 2014; 346: 256-259. [7] Gulley ML, Pulitzer DR, Eagan PA, Schneider BG. Epstein-Barr virus infection is an early event in gastric carcinogenesis and is independent of bcl-2 expression and p53 accumulation. Hum Pathol 1996; 27: 20-27. [8] Ojima H, Fukuda T, Nakajima T, Nagamachi Y. Infrequent overexpression of P53 protein in Epstein-Barr virus-associated gastric carcinomas. Jpn J Cancer Res 1997; 88: 262-266. [9] Moritani S, Sugihara H, Kushima R, Hattori T. Different roles of p53 between Epstein-Barr virus-positive and -negative gastric carcinomas of matched histology. Virchows Arch 2002; 440: 367-375. [10] Ushiku T, Chong JM, Uozaki H, Hino R, Chang MS, Sudo M, Rani BR, Sakuma K, Nagai H, Fukayama M. p73 gene promoter methylation in Epstein-Barr virus-associated gastric carcinoma. Int J Cancer 2007; 120: 60-66.
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[11] Lee JH, Kim SH, Han SH, An JS, Lee ES, Kim YS. Clinicopathological and molecular characteristics of Epstein-Barr virus-associated gastric carcinoma: a meta-analysis. J Gastroenterol Hepatol 2009; 24: 354-365. [12] Leung SY, Chau KY, Yuen ST, Chu KM, Branicki FJ, Chung LP. P53 overexpression is different in Epstein-Barr virus-associated and EpsteinBarr virus-negative carcinoma. Histopathology 1998; 33: 311-317. [13] Herath CH, Chetty R. Epstein-Barr virus-associated lymphoepithelioma-like gastric carcinoma. Arch Pathol Lab Med 2008; 132: 706709. [14] Lima VP, de Lima MA, André AR, Ferreira MV, Barros MA, Rabenhorst SH. H pylori (CagA) and Epstein-Barr virus infection in gastric carcinomas: correlation with p53 mutation and c-Myc, Bcl-2 and Bax expression. World J Gastroenterol 2008; 14: 884-891. [15] Matsunou H, Konishi F, Hori H, Ikeda T, Sasaki K, Hirose Y, Yamamichi N. Characteristics of Epstein-Barr virus-associated gastric carcinoma with lymphoid stroma in Japan. Cancer 1996; 77: 1998-2004. [16] Conticello SG. The AID/APOBEC family of nucleic acid mutators. Genome Biol 2008; 9: 229. [17] Vieira VC, Soares MA. The role of cytidine deaminases on innate immune responses against human viral infections. Biomed Res Int 2013; 2013: 683095. [18] Shimizu T, Marusawa H, Endo Y, Chiba T. Inflammation-mediated genomic instability: roles of activation-induced cytidine deaminase in carcinogenesis. Cancer Sci 2012; 103: 1201-1206. [19] Marusyk A, Tabassum DP, Altrock PM, Almendro V, Michor F, Polyak K. Non-cell-autonomous driving of tumour growth supports sub-clonal heterogeneity. Nature 2014; 514: 54-58.
Int J Clin Exp Pathol 2015;8(9):11766-11771
Case Report A case of gastric cancer with heterogeneous components of EB virus (+)/TP53 (+) and EB virus (-)/TP53 (-) Ikuo Matsuda1, Kazuomi Kan2, Sadayuki Doi2, Yoshiyuki Motoki2, Masayuki Onodera3, Seiichi Hirota1 1 3
Department of Surgical Pathology, Hyogo, Japan; 2Department of Surgery, Kawanishi City Hospital, Hyogo, Japan; Department of Pathology, Kawanishi City Hospital, Hyogo, Japan
Received July 9, 2015; Accepted August 21, 2015; Epub September 1, 2015; Published September 15, 2015 Abstract: Epstein-Barr virus (EBV)-associated gastric adenocarcinoma is a histological subtype of gastric adenocarcinoma, in which all of the carcinoma cells are basically positive for EBV-encoded small RNA (EBER) by in situ hybridization. Although its typical histology has some overlap with gastric carcinoma with lymphoid stroma, absence of massive lymphoid infiltrate is sometimes observed either in whole or in part. EBV-associated adenocarcinoma is one of the four representative molecular pathological subtypes recently identified by comprehensive genomic analysis of gastric adenocarcinomas. According to the analysis, typical EBV-associated gastric adenocarcinoma constitutes an independent molecular pathological subgroup, which is mutually exclusive to TP53-mutated adenocarcinoma with chromosomal instability, another molecular pathological subtype in gastric adenocarcinomas. Here, we report a rare case of gastric cancer heterogeneously composed of EBER (+)/TP53 (+) and EBER (-)/TP53 (-) portions. The EBER (+)/TP53 (+) component with massive lymphoid infiltrate surrounded the EBER (-)/TP53 (-) component showing well to moderately differentiated tubular adenocarcinoma. Although collision of two independent gastric cancers could be the simplest and most possible explanation for this situation, we discussed another possibility. In the case of gastric collision tumors, concurrent development of EBER (+) gastric adenocarcinomas and EBER (-) gastric adenocarcinomas in a single stomach is a rare incident. Since presence of the EBER (+)/TP53 (+) tumor component is atypical in itself, we also discussed the mechanism of development of the clone. Keywords: Gastric adenocarcinoma, Epstein-Barr virus, adenocarcinoma with lymphoid stroma, TP53, collision tumor
Epstein-Barr virus (EBV)-associated gastric adenocarcinoma is a histological subtype of gastric adenocarcinoma, in which all of the carcinoma cells are basically positive for EBVencoded small RNA (EBER) by in situ hybridization [1]. Therefore, it is reasonable to assume that the EBV infection occurs at the early stage of the tumorigenesis and may be related to its initiation. Although its typical histology has some overlap with gastric carcinoma with lymphoid stroma, absence of massive lymphoid infiltrate is sometimes observed either in whole or in part. More than 80% of cases of gastric carcinoma with lymphoid stroma are associated with Epstein-Barr virus (EBV) infection [2].
molecular pathological subtypes [3]. The first subtype is EBV-associated adenocarcinoma, the second one is associated with microsatellite instability characterized by loss of expression of MLH1 protein, the third one is diffuseproliferating and poorly-differentiated histological subtype characterized by mutations in RhoA signaling pathway, and the last one is intestinal subtype often associated with chromosomal instability and TP53 mutation [3]. The subtype with MLH1 loss is often associated with CpG island methylator phenotype [3]. It was reported that these four subtypes are mutually exclusive in general [3]. Therefore, EBER mRNA positivity concurrent with diffuse TP53 protein positivity within a tumor is considered to be an exception rather than a rule.
A recent comprehensive genomic analysis of gastric adenocarcinoma identified at least four
Histologically, it is common that mixture of different patterns composes a tumor. At cellular
Introduction
Gastric cancer with EBV (+)/TP53 (+) and EBV (-)/TP53 (-) components TP53 (+) component in the peripheral zone surrounded EBER (-)/TP53 (-) component in the central zone. Although we considered that collision of two independent gastric cancers could be the most possible explanation for this situation, we discussed another possibility. Since EBER (+)/TP53 (+) tumor component in this tumor is atypical in itself, we discussed mechanism for this phenomenon. Case report
Figure 1. Loupe view of the resected tumor reconstructed by two virtual slides. (A) Hematoxylin-eosin (HE) stain. (B) In situ hybridization of Epstein-Barr virus-encoded small RNAs (EBER). (C) TP53 immunohistochemistry. The area of the whole tumor is outlined by a dotted curve in (A). In (B) and (C), positive cells are stained brown. The intratumor heterogeneity was illustrated by in situ hybridization of EBER (B) and immunohistochemistry for TP53 (C). That is, the tumor cells in the peripheral zone, demarcated by a solid curve in each figure, were positive for EBER (B) as well as TP53 (C). In contrast, the tumor cells in the central zone, which is the residual portion of the tumor and is demarcated by dotted and solid lines, were basically negative for EBER (B) and TP53 (C). Both of the two zones were homogeneously positive for MLH1 immunohistochemistry (data not shown). Bar: 5 mm.
level, moreover, tumors are composed of assembly of multiple heterogeneous clones. The so-called intratumor heterogeneity has been recently refined as genomic heterogeneity among the tumor cells, revealed by analysis using next generation sequencing [4-6]. The case of gastric adenocarcinoma is no exception. In the routine diagnostic histopathology, the correlation between morphological heterogeneity and genomic one in the tumor is of clinical significance. Morphological heterogeneity of the tumor may sometimes provide an important clue to genomic heterogeneity of the tumor, which is often associated with responses to particular molecular-targeted therapy. A collision tumor, in which more than two independent tumors collide to constitute a tumor, might be one of the extreme presentations of intratumor heterogeneity. Here we report a rare case of gastric cancer heterogeneously composed of EBER (+)/TP53 (+) and EBER (-)/TP53 (-) portions. EBER (+)/
11767
A 70-year-old Japanese man was admitted to Kawanishi City Hospital for examination of progressive anemia. At the age of 58, he had suffered from acute myocardial infarction, for which coronary stenting was performed, and intrahepatic cholangiocellular carcinoma, which was completely resected by partial hepatectomy. He had also been followed for type 2 diabetes mellitus and chronic hepatitis C. Blood test showed that his hemoglobin had decreased from 13.1 to 11.1 g/dL (normal range: 13.217.2) during 4 months. Gastrointestinal bleeding was suspected. Endoscopic examination of the gastrointestinal tract revealed Borrmann type 2 tumor of around 2.5 cm in diameter in the posterior wall of the gastric corpus (data not shown). The biopsy of the tumor showed the proliferation of tubular adenocarcinoma (data not shown). Pylorus-preserving partial gastrectomy was performed. The HE image of the resected specimen showed that the tumor was saucer-shaped at low power view (Figure 1A), which was composed of the central portion and the peripheral one. The deepest portion of the invasion reached to the superficial layer of the muscularis propria (Figure 1A). The central portion of the tumor mainly consisted of tubular proliferation of atypical epithelial cells, which was diagnosed as tubular adenocarcinoma of well to moderately differentiated type (Figure 2A). In contrast, peripheral portion consisted of tumor cells proliferating diffusely and sparsely, forming small aggregates or nests, which was diagnosed as poorly differentiated adenocarcinoma (Figure 2C). The morphological distinction between the central and peripheral components of the tumor was clearly highlighted with immunohistochemistry of pankeratin AE1/AE3 (Figures 2B, 2D, 3B). In addition to the morphological differences, the peripheral portion of
Int J Clin Exp Pathol 2015;8(9):11766-11771
Gastric cancer with EBV (+)/TP53 (+) and EBV (-)/TP53 (-) components
Figure 2. Representative histology of the resected tumor in the central and peripheral zones. (A, B) Central zone. (C, D) Peripheral zone. (A, C) HE stain. (B, D) Immunohistochemistry for pankeratin AE1/AE3. The tumor cells formed tubular structure in the central zone (A, B), while they proliferated diffusely in nested or isolated clusters intermingled with lymphoid infiltrate in the peripheral zone (C, D). Original magnification: × 200. Bar: 100 μm.
the tumor was intermingled with heavy lymphoid infiltrate, which obscured the distinction between the tumor foci and inflammatory cells (Figure 2C). This so-called lymphoepithelial lesion was reminiscent of EBV-associated carcinoma such as nasopharyngeal carcinoma. In order to examine a possibility of EBV-associated gastric adenocarcinoma, in situ hybridization of EBER was performed. Unexpectedly, the tumor cells were heterogeneously positive for EBER; that is, the central and tubular portion of the tumor was negative for EBER, while the peripheral and poorly differentiated portion of the tumor was positive (Figures 1B and 3C). Further immunohistochemical examination of the tumor showed that the central component of the tumor was almost negative for TP53 except for sparsely distributed positive cells in the mucosal surface, while the peripheral component was diffusely positive for TP53 (Figures 1C and 3D). Both the central and peripheral 11768
portions of the tumor were positively stained for MLH1 immunohistochemistry (Figure 3E). Thus, the central zone of the tumor was composed of EBER (-)/TP53 (-)/MLH1 (+) cells, while the peripheral zone of the tumor was composed of EBER (+)/TP53 (+)/MLH1 (+) cells (Figure 3C-E). Discussion In this paper, we report a case of gastric adenocarcinoma composed of two heterogeneous portions with clear borderline formation: the central core zone and the peripheral zone. The central zone was composed of EBER (-)/TP53 (-)/MLH1 (+) tumor cells. In contrast, the peripheral zone was composed of EBER (+)/TP53 (+)/ MLH1 (+) tumor cells, surrounding the central core zone. A recent comprehensive genomic analysis revealed that gastric adenocarcinomas are comInt J Clin Exp Pathol 2015;8(9):11766-11771
Gastric cancer with EBV (+)/TP53 (+) and EBV (-)/TP53 (-) components
Figure 3. High power view of the resected tumor at the borderline between the central and peripheral zones described in Figure 1. (A) HE stain. (B, D, E) Immunohistochemistry. (C) In situ hybridization. (B) Pankeratin AE1/AE3. (C) EBER. (D) TP53. (E) MLH1. In each figure, the central zone is in the left half side of the solid line, while the peripheral zone is in the right half side. In (B) to (E), positive cells are stained brown. All of the tumor cells were highlighted by AE1/AE3 immunohistochemistry (B). The central zone of the tumor in the left half of each figure was negative for EBER (C) and TP53 (D), and positive for MLH1 (E). On the other hand, the peripheral zone of the tumor in the right half of each figure was positive for EBER (C) and TP53 (D), as well as MLH1 (E). Original magnification: × 200. Bar: 100 μm.
posed of at least four molecular pathological subtypes, each characterized by association with either EBV, loss of MLH1 protein, TP53 mutation, or mutations in RhoA pathway [3]. It was reported that these four subtypes are mutually exclusive in general [3]. Consistent with this, there were several reports suggesting that EBV-associated gastric adenocarcinomas do not harbor TP53 mutations in most cases [7-11], although some discrepant results were also reported [12-14]. In this regard, the EBER (+)/TP53 (+) tumor component in the peripheral zone appears to be atypical for EBV-associated gastric adenocarcinoma. The most straightforward explanation for the present case might be a collision tumor composed of two independent gastric adenocarcinomas, one of which is EBER (+)/TP53 (+) while the other is EBER (-)/TP53 (-). EBV-associated gastric adenocarcinomas are reported to be associated with synchronous or metachronous independent adenocarcnomas in the stomach, and Matsunou et al. [15] reported that 22 out of 26 cases (86.4%) of gastric adenocarcinomas with lymphoid stroma which developed synchronously or metachronously in patients 11769
with gastric adenocarcinomas with lymphoid stroma were EBER-positive. They also described that all of 4 gastric adenocarcinomas without lymphoid stroma which developed synchronously or metachronously in patients with gastric adenocarcinomas with lymphoid stroma were EBER-positive as well [15]. Since these data indicate that concurrent development of EBER (-) gastric adenocarcinomas is a rare incident in the stomach with EBER (+) gastric adenocarcinomas, collision of EBER (+) adenocarcinoma with EBER (-) adenocarcinoma in our case might be an extraordinary event. As another possibility for the present case, one of the two zones may be a metastasis of other primary tumor to the original gastric cancer. Since the patient had undergone partial hepatectomy for cholangiocellular carcinoma 12 years before, the EBER (-)/TP53 (-)/MLH1 (+) zone could be the metastatic recurrence of the cholangiocellular carcinoma. Although the cholangiocellular carcinoma was also shown to be EBER (-)/TP53 (-)/MLH1 (+) (data not shown), CK7-positivity of the cholangiocellular carcinoma was not consistent with CK7-negativity of the present gastric cancer (data not shown). Int J Clin Exp Pathol 2015;8(9):11766-11771
Gastric cancer with EBV (+)/TP53 (+) and EBV (-)/TP53 (-) components How did TP53-immunopositivity evolved in EBER (+) tumor cells in the peripheral zone? A candidate cell-autonomous link, if any, between EBV infection and TP53 mutation may be the expression of apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) family proteins in the tumor cells [5, 16]. As a mechanism of host immunity to virus, APOBEC family restricts replication of viral genomes (for example, EBV genome) by enzymatically introducing cytidine to thymine conversion in the DNA [17]. This may not be achieved without the cost of potential introduction of mutations into the host genome, presumably including TP53 mutation. A recent study by next generation sequencing showed that intratumor heterogeneity is partly generated by an APOBEC-mediated mechanism [5]. Expression of activation-induced cytidine deaminase (AID), another APOBEC family, in gastric adenocarcinoma was also reported [18]. AID may be an APOBEC family involved in TP53 mutation in Helicobacter pylori-mediated tumorigenesis of the stomach [18]. The borderline formation between the two components in the present case can be easily explained by a cell competition mechanism at the junction of the two. A typical example of cell competition may be observed in senescenceassociated secretory phenotype (SASP) in aging, where senescent “loser” subclones cease to proliferate while the surrounding “winner” cells proliferate in response to cytokines secreted by the senescent “loser” subclones. It is tempting to speculate that cytokine secretion, presumably associated with inflammatory infiltration in EBV-positive cancer, may play a role in this borderline formation, just as in SASP. The importance of non-cell-autonomous driving of tumor growth and clonal heterogeneity has recently been experimentally substantiated [19]. In conclusion, we report a rare case of gastric cancer, in which EBER (-)/TP53 (-) component in the central zone and EBER (+)/TP53 (+) one in the peripheral zone are coexistent. A collision tumor of two independent gastric cancers could be the most possible explanation for this situation. Concurrent development of EBER (+) gastric adenocarcinoma and EBER (-) gastric adenocarcinoma in a single stomach is a rare incident. Moreover, EBER (+)/TP53 (+) tumor
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component in this tumor is considered to be atypical in itself for EBV-associated gastric adenocarcinoma. We speculate that the sharp borderline formation of two components in the present case may be promoted by cell-autonomous as well as non-cell-autonomous mechanisms, both of which may be related to EBV infection. Acknowledgements We thank all the colleagues in the Department of Surgical Pathology, Hyogo College of Medicine, and Department of Pathology, Kawanishi City Hospital, for preparation of pathological specimen. This work was partly supported by Grant-in-Aid for Researchers, Hyogo College of Medicine, 2014. Disclosure of conflict of interest None. Address correspondence to: Dr. Seiichi Hirota, Department of Surgical Pathology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 6638501, Japan. Tel: +81798456666; Fax: +81798456671; E-mail: [email protected]
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